Ly6g antibody

Formalin-fixed tissue samples were embedded in paraffin, and 5 μm sections were cut. Sections were stained with hematoxylin and eosin (H&E staining) for the evaluation of necrosis. Neutrophils and γH2AX positive cells were detected using a Ly6G antibody (1:400, Servicebio, Wuhan, China) and a γH2AX antibody (1:500, Huabio, Hangzhou, China) respectively. H&E staining and immunohistochemistry (IHC) analysis were evaluated by microscopy, and five visual fields were randomly selected for observation. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by an automatic biochemical analyzer from Rayto Life and Analytical Sciences Co., Ltd. (Shenzhen, China).

Mouse liver tissues were embedded in an optimal cutting temperature (OCT) compound (Servicebio) to make frozen sections. Serial sections of the tissues of 8 μm thickness were cut and used for the fluorescence observations. Double immunofluorescent staining was performed to evaluate immune cell infiltration with Ly6G antibody (Servicebio) and F4/80 antibody (Servicebio). The apoptosis of hepatocytes was detected using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay from a commercial kit (Yeasen), following the manufacturer’s instructions. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China), and the positive cells were visualized using a fluorescence microscope.

Neutrophils were incubated with DiR-labeled liposomes for 2 h, followed by washing with PBS to remove the free liposomes. Mice were infected intranasally with 4 × 10⁶ CFU of PA14 as aforementioned. 2 h post infection, the neutrophils were injected intravenously into the tail vein. 2 h after injection of the cells, the lungs were isolated and fixed by formaldehyde. The cryosections of the lungs were stained with a Ly6G antibody (Servicebio, Wuhan, China) and DAPI (Servicebio, Wuhan, China) and observed by microscopy.